Differential utilization of carbon substrates by aggregate-associated and water-associated heterotrophic bacterial communities

The capacity to utilize carbon substrates is fundamental to the functioning of heterotrophic microbial communities in aquatic environments. Carbon-source utilization within the water column, however, is not a bulk property because microbial communities are patchily distributed on suspended organic aggregates (i.e., marine snow, marine aggregates, river aggregates, organic detritus, and bioflocs). In this study, Biolog Ecoplates were used to evaluate the metabolic capacity of heterotrophic bacterial communities associated with aggregates compared to communities in the surrounding water. Overall, aggregate-associated microbial communities demonstrated higher levels of metabolism, metabolic versatility, and functional redundancy, and a more consistent pattern of carbon-source utilization compared with water-associated communities. In addition, aggregate-associated communities more effectively exploited available resources, including representatives from several biochemical guilds and nitrogen-containing carbon sources. Within the aggregate-associated microbial community, metabolic activity was significantly higher in the presence of polymers, amino acids, and carbohydrates relative to amines and carboxylic acids. In comparison, metabolic activity of water-associated communities exceeded a threshold value for only two of the five guilds (polymers and carbohydrates) evaluated. These results suggest that compared with their free-living counterparts, aggregate-associated communities have a greater capacity to respond to a wider array of carbon inputs. Results also underscore the importance of targeting organic aggregates to better understand the role of microbial processes in ecosystem functioning.     

Superficial and deeper layers of dog normal articular cartilage. Role of hyaluronate and link protein in determining the sedimentation coefficients distribution of the nondissociatively extracted proteoglycans

Proteoglycans were extracted under nondissociative conditions from superficial and deeper layers of dog normal articular cartilage. The purified a-A1 preparations were characterized by velocity gradient centrifugation. Superficial specimens exhibited an abundant population of slow sedimenting aggregates whereas the aggregates of deeper preparations sedimented as two well-defined families of molecules. These dissimilarities in the size distribution of the aggregates observed between superficial and deeper a-A1 preparations derived most of all from differences in their content of hyaluronate and link proteins: (a) superficial preparations contained twice as much hyaluronate as deeper specimens; (b) superficial aggregates were link-free and unstable at pH 5.0 whereas deeper preparations contained link-proteins and their faster sedimenting aggregates were stabilized against dissociation at pH 5.0. In these proteoglycan preparations from different cartilage layers, the monomers exhibited an identical capacity for aggregation and the hyaluronate molecules displayed quite similar molecular weight (Mr = 5 x 10(5] and aggregating capacity. These observations as well as aggregating studies conducted with highly purified link protein and purified hyaluronate specimens of different molecular weights support the following conclusions: (a) link protein not only stabilizes proteoglycan aggregates but also enhances the aggregating capacity of hyaluronate; (b) for all practical purposes, the slow sedimenting aggregates represent a secondary complex of hyaluronate and proteoglycan monomers whereas the fast sedimenting aggregates may be considered as a ternary complex wherein link protein stabilizes the hyaluronate-proteoglycans interaction; (c) the distinctive heterogeneity of articular cartilage can be related to structurally different proteoglycan aggregates. The structural dissimilarities observed between superficial and deeper aggregates could reflect the different macromolecular organization of the proteoglycan molecules in the territorial and interterritorial matrices, respectively.     

Changes in erythrocyte and thrombocyte aggregation after ultraviolet irradiation

Lipid peroxidation which occurs in blood serum under ultraviolet irradiation was studied. The products of these reaction suppress ADP-induced aggregation of native platelets. The rouleaux-forming capacity increased after UV-irradiation of plasma and serum albumin. Under UV-irradiation the aggregates of albumin molecules are supposed to form the aggregates of albumin molecules which bind the erythrocytes in rouleaux.     

Aggregates of nisin with various bactoprenol-containing cell wall precursors differ in size and membrane permeation capacity

Many lantibiotics use the membrane bound cell wall precursor Lipid II as a specific target for killing Gram-positive bacteria. Binding of Lipid II usually impedes cell wall biosynthesis, however, some elongated lantibiotics such as nisin, use Lipid II also as a docking molecule for pore formation in bacterial membranes. Although the unique nisin pore formation can be analyzed in Lipid II-doped vesicles, mechanistic details remain elusive. We used optical sectioning microscopy to directly visualize the interaction of fluorescently labeled nisin with membranes of giant unilamellar vesicles containing Lipid II and its various bactoprenol precursors. We quantitatively analyzed the binding and permeation capacity of nisin when applied at nanomolar concentrations. Specific interactions with Lipid I, Lipid II and bactoprenol-diphosphate (C₅₅-PP), but not bactoprenol-phosphate (C₅₅-P), resulted in the formation of large molecular aggregates. For Lipid II, we demonstrated the presence of both nisin and Lipid II in these aggregates. Membrane permeation induced by nisin was observed in the presence of Lipid I and Lipid II, but not in the presence of C₅₅-PP. Notably, the size of the C₅₅-PP–nisin aggregates was significantly smaller than that of the aggregates formed with Lipid I and Lipid II. We conclude that the membrane permeation capacity of nisin is determined by the size of the bactoprenol-containing aggregates in the membrane. Notably, transmitted light images indicated that the formation of large aggregates led to a pinch-off of small vesicles, a mechanism, which probably limits the growth of aggregates and induces membrane leakage.     

耕作方式对潮土土壤团聚体微生物群落结构的影响

为探究不同耕作方式对潮土土壤团聚体微生物群落结构和多样性的影响,采用磷脂脂肪酸(PLFA)法测定了土壤团聚体中微生物群落。试验设置4个耕作处理,分别为旋耕+秸秆还田(RT)、深耕+秸秆还田(DP)、深松+秸秆还田(SS)和免耕+秸秆还田(NT)。结果表明:与RT相比,DP处理显著提高了原状土壤和>5 mm粒级土壤团聚体中真菌PLFAs量和真菌/细菌,为真菌的繁殖提供了有利条件,有助于土壤有机质的贮存,提高了土壤生态系统的缓冲能力;提高了5～2 mm粒级土壤团聚体中细菌PLFAs量,降低了土壤革兰氏阳性菌/革兰氏阴性菌,改善了土壤营养状况;提高了<0.25 mm粒级土壤团聚体中微生物丰富度指数。总的来说,深耕+秸秆还田(DP)对土壤团聚体细菌和真菌生物量有一定的提高作用,并且在一定程度上改善了土壤团聚体微生物群落结构,有利于增加土壤固碳能力和保持土壤微生物多样性。冗余分析结果表明,土壤团聚体总PLFAs量、细菌、革兰氏阴性菌和放线菌PLFAs量与土壤有机碳相关性较强,革兰氏阳性菌PLFAs量与总氮相关性较强。各处理较大粒级土壤团聚体微生物群落主要受碳氮比、含水量、pH值和团聚体质量分数的影响,较小粒级土壤团聚体微生物群落则主要受土壤有机碳和总氮的影响。     

Stage-related capacity for limb chondrogenesis in cell culture.

Cells from wing buds of varying-stage chick embryos were dissociated and grown in culture to test their capacity for cartilage differentiation. Micro-mass cultures were initiated with a cell layer greater than confluency, which occupied a restricted area of the culture dish surface (10–13 mm²). Cells from stage 24 chick embryo wing buds (prior to the appearance of cartilage in vivo) undergo cartilage differentiation in such cultures. Typically, during the first 1–2 days of culture, cells form aggregates (clusters of cells with a density 1.5 times greater than that of the surrounding nonaggregate area). By Day 3, virtually all aggregates differentiate into cartilage nodules which are easily recognized by their Alcian blue staining (pH 1.0) extracellular matrix. Subsequently, nodules increase in size, and adjacent nodules begin to coalesce. Micro-mass cultures were used to test the chondrogenic capacity of wing bud cells from chick embryos representing the different stages of limb development up to the appearance of cartilage in vivo (stages 17–25). Cells from embryo stages 21–24 form aggregates which differentiate into cartilage nodules in vitro with equal capacity (scored as number of nodules per culture). In contrast, cells from embryo stages 17–19 form aggregates in similar numbers, but these aggregates never differentiate into nodules under routine conditions. However, aggregates which form in cultures of stage 19 wing bud cells do differentiate into cartilage nodules if exposed to dibutyryl cyclic AMP and theophylline. Cells from stage 20 embryos manifest a varying capacity to form cartilage nodules; apparently, this is a transition stage. Cells from stage 25 embryos produce cartilage in vitro without forming either aggregates or nodules. Based on the results presented in this paper, the authors propose a model for cartilage differentiation from embryonic mesoderm cells involving: (1) aggregation, (2) acquisition of the ability to respond to the environment in the aggregate, (3) elevated intracellular cyclic AMP levels, and (4) stabilization and expression of cartilage phenotype.     

Rapid production of wheat cell suspension cultures directly from immature embryos

The aim of this study was to produce suspension cultures of winter wheat directly from immature embryos bypassing the callus stage, and to determine their capacity for growth and regeneration in comparison to suspension cultures produced from callus. The study was carried out using Polish winter wheat varieties: ‘Grana’ and ‘Rosa’. Immature embryos were isolated, homogenized and transferred directly to liquid medium supplemented with 2,4-D. Actively dividing cell cultures were obtained within 2 months after the cultures were started. Suspension cultures from callus of immature embryos was also produced. With both cultivars, faster growth was observed in the suspension cultures produced directly from embryos than in the suspensions produced from callus. Metabolic activity was higher in the suspension culture produced directly from embryos than in the suspension derived from callus only in ‘Grana’. The production of 1-amiocyclopropane-1-carboxylic acid (ACC), an ethylene precursor, was lower in the suspension cultures produced directly from embryos than in the suspensions produced from callus. Morphogenic capacity was significantly higher in aggregates derived directly from embryos than in aggregates derived from callus. With ‘Rosa’, about one third of the aggregates derived directly from embryos regenerated shoots. Production of ACC was lower in ‘Rosa’ cell culture that regenerated then in other cell cultures that did not. Photosystem II reactions were more efficient in dark green aggregates than in light green or pale green aggregates which were unable to regenerate. With the method presented, wheat cell suspension cultures with a regeneration potential can be produced in 2 or 3 months less time than with traditional methods.     

Low-density lipoproteins with different capacity for aggregation

Preparations of low-density lipoproteins from healthy donor blood contain lipoprotein particles with different capacity for aggregation: upon stirring, some particles form aggregates significantly more quick than others. After stirring, lipoprotein particles are separated by ultracentrifugation into two fractions: a fraction of large aggregates and a fraction of small particles without intermediate forms. It is known that lipoprotein aggregates can accelerate intracellular accumulation of lipids. Therefore, it is supposed that particles of high aggregation ability are more atherogenic.     

Assembly of proteoglycan aggregates in cultures of chondrocytes from bovine tracheal cartilage.

The assembly of proteoglycan aggregates in chondrocyte cell cultures was examined in pulse-chase experiments with the use of [35S]sulphate for labelling. Rate-zonal centrifugation in linear sucrose density gradients (10-50%, w/v) was used to separate the aggregated proteoglycans from monomers and to assess the size of the newly formed aggregates. The proportion of aggregates stabilized by link protein was assessed by competition with added exogenous aggregate components. The capacity of the proteoglycans synthesized in culture to compete with exogenous nasal-cartilage proteoglycans for binding was studied in dissociation-reassociation experiments. The results were as follows. (a) The proteoglycan monomers and the hyaluronic acid are exported separately and combined extracellularly. (b) The size of the aggregates increases gradually with time as the proportion of monomers bound to hyaluronic acid increases. (c) All of the aggregates present at a particular time appear to be link-stabilized and therefore not dissociated by added excess of nasal-cartilage proteoglycan monomer or hyaluronic acid oligomers. (d) The free monomer is apparently present as a complex with link protein. The monomer-link complexes are then aggregated to the hyaluronic acid. (e) The aggregates synthesized in vitro and the nasal-cartilage aggregates differ when tested for link-stabilization by incubation at low pH. The aggregates synthesized in vitro were completely dissociated whereas the cartilage proteoglycans remained aggregated. The results obtained from dissociation-reassociation experiments performed at low pH indicate that the proteoglycan monomer synthesized in vitro does not bind the hyaluronic acid or the link protein as strongly as does the nasal-cartilage monomer.     

Phenotypes of non-attached Pseudomonas aeruginosa aggregates resemble surface attached biofilm

For a chronic infection to be established, bacteria must be able to cope with hostile conditions such as low iron levels, oxidative stress, and clearance by the host defense, as well as antibiotic treatment. It is generally accepted that biofilm formation facilitates tolerance to these adverse conditions. However, microscopic investigations of samples isolated from sites of chronic infections seem to suggest that some bacteria do not need to be attached to surfaces in order to establish chronic infections. In this study we employed scanning electron microscopy, confocal laser scanning microscopy, RT-PCR as well as traditional culturing techniques to study the properties of Pseudomonas aeruginosa aggregates. We found that non-attached aggregates from stationary-phase cultures have comparable growth rates to surface attached biofilms. The growth rate estimations indicated that, independently of age, both aggregates and flow-cell biofilm had the same slow growth rate as a stationary phase shaking cultures. Internal structures of the aggregates matrix components and their capacity to survive otherwise lethal treatments with antibiotics (referred to as tolerance) and resistance to phagocytes were also found to be strikingly similar to flow-cell biofilms. Our data indicate that the tolerance of both biofilms and non-attached aggregates towards antibiotics is reversible by physical disruption. We provide evidence that the antibiotic tolerance is likely to be dependent on both the physiological states of the aggregates and particular matrix components. Bacterial surface-attachment and subsequent biofilm formation are considered hallmarks of the capacity of microbes to cause persistent infections. We have observed non-attached aggregates in the lungs of cystic fibrosis patients; otitis media; soft tissue fillers and non-healing wounds, and we propose that aggregated cells exhibit enhanced survival in the hostile host environment, compared with non-aggregated bacterial populations.     

Colonization of Wheat Root Hairs and Roots by Agrobacteria

Formation of extracellular structures in pure culture and in interaction with wheat root surface was studied by scanning and transmission electron microscopy. The effects of various factors (growth temperature as well as pretreatment of agrobacteria with kalanchoe extract, acetosyringone, and centrifugation) on formation of extracellular structures was tested. The data on Agrobacterium tumefaciens (wild-type strain C58 and mutants LBA2525 (virB2::lacZ) and LBA288 (without the Ti plasmid)) adhesion to wheat root surface and root hairs after pretreatment of agrobacteria with inducer of virulence genes (vir) acetosyringone were obtained. Formation of agrobacterial cell aggregates on wheat root hair tips was demonstrated. The proportion of root hairs with agrobacterial aggregates on the root hair tip insignificantly changed after pretreatment with acetosyringone but considerably increased after treatment of A. tumefaciens C58 and LBA2525 with kalanchoe leaf extract. The most active colonization of root hairs and formation of agrobacterial aggregates on hair root tips was observed at 22°C. The capacity of agrobacteria for adhesion on monocotyledon surface could be changed by pretreatment of bacteria with various surface-active substances. Bacterial cells subjected to centrifugation had a decreased capacity for attachment to both wheat root surface and root hairs. The relationship between the capacity for adhesion and pilus production in agrobacteria was considered.     

宁南山区植被恢复对土壤团聚体养分特征及微生物特性的影响

测定了宁夏黄土丘陵区植被恢复近30年的天然草地和农地不同粒径团聚体的土壤养分含量、微生物生物量、呼吸特性和生态化学计量比等指标,探索黄土丘陵区植被恢复对不同粒径土壤团聚体的养分特性和微生物学性质的影响.结果表明: 微团聚体(粒径<0.25 mm)质量百分比、各粒径土壤团聚体养分(有机碳、全氮、速效钾)含量、C/N均表现为天然草地大于农地,其中1～2 mm粒径团聚体有机碳、全氮含量在天然草地和农地中均最高,C/N也较高,说明植被恢复能有效促进土壤团粒的形成,适宜养分积累和有机碳的汇集,且在1～2 mm粒径团聚体上表现最为突出;天然草地各粒径土壤团聚体微生物生物量(碳、氮)、基础呼吸强度均高于农地,而呼吸熵低于农地,可见植被恢复措施可有效提高各粒径土壤微生物生物量与活性,并使土壤生境趋于稳定;但由于养分特性的差异,不同粒径团聚体微生物特性对植被修复的响应存在差异,其中天然草地土壤1～2 mm粒径团聚体微生物生物量碳,<0.25、0.25～1、1～2 mm粒径团聚体微生物生物量氮,以及1～2、>5 mm粒径团聚体基础呼吸强度显著高于其他粒径,即上述粒径团聚体的微生物生物量和微生物活性在植被恢复过程中逐渐被改善.表明宁南山区植被恢复有效改善了土壤团聚体的肥力状况与结构特征,且1～2 mm粒径团聚体的改良效果最为突出.     

The assembly of early components of complement on antibody-antigen aggregates and on antibody-coated erythrocytes.

Radioimmune assays were developed to assay the binding of complement components C1q, C1s and C4 to antibody aggregates and to cell-bound antibody. The binding of the components was compared with the haemolytic activity and with the capacity to form the C3 convertase activity in the presence of excess C2. The destruction of whole complement and of C4 activity is similar per 1,000 molecules of antibody in aggregates and cell-bound antibody, as is the binding of C1g and C1s, the latter being in a 1:2 molar ratio. The binding of C4 is about 12 times greater, per 1,000 molecules of antibody, on cells than in aggregates. However, the effective C4 molecules, as judged by the formation of C3 convertase activity, are much more similar on cells and aggregates. An assembly mechanism of the early components of complement on antibody-coated cells, which is compatible with these results, is suggested.     

Cytosolic and nuclear aggregation of the amyloid beta-peptide following its expression in the endoplasmic reticulum

Misfolded secretory and membrane proteins are known to be exported from the endoplasmic reticulum (ER) to the cytosol where they are degraded by proteasomes. When the amount of exported misfolded proteins exceeds the capacity of this degradation mechanism the proteins accumulate in the form of pericentriolar aggregates called aggresomes. Here, we show that the amyloid beta-peptide (Abeta) forms cytosolic aggregates after its export from the ER. These aggregates share several constituents with aggresomes. However, Abeta aggregates are distinct from aggresomes in that they do not accumulate around the centrosome but are distributed randomly around the nucleus. In addition to these cytosolic aggregates, Abeta forms intranuclear aggregates which have as yet not been found for proteins exported from the ER. These findings show that proteins exported from the ER to the cytosol which escape degradation by the proteasome are not necessarily incorporated into aggresomes. We conclude that several distinct aggregation pathways may exist for proteins exported from the ER to the cytosol.     

Thalidomide may inhibit proliferation of mesenchyme in human limb buds

Limb buds from 4- and 4.5-week-old human embryos were cultured on agar medium consisting of Medium 199, chick embryo extract and horse serum for 4 days with or without thalidomide (1-1.5 microgram/ml), and the direct effect of thalidomide was examined morphologically in histological preparations. In the explants treated with thalidomide, mitotic figures of mesenchymal cells were significantly decreased both in overall explant and in mesenchymal cell aggregates, but the extracellular matrix in the mesenchymal cell aggregates was seen in the experimental and control explants. These findings suggest that thalidomide affects undifferentiated and differentiated mesenchymal cell proliferation but not the chondrogenic capacity of the mesenchyme.     

Peroxyl-oxidized erythrocyte membrane band 3 protein with anion transport capacity is degraded by membrane-bound proteinase

Human red blood cells anion exchange protein (band 3) exposed to peroxyl radicals produced by thermolysis of 2,2'-azo-bis(2-amidinopropane) (AAPH) is degraded by proteinases that prevent accumulation of oxidatively damaged proteins. To assess whether this degradation affects anion transport capacity we used the anionic fluorescent probe 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-y) amino] ethanosulfonate (NBD-taurine). A decrease of band 3 function was observed after exposure to peroxyl radicals. In the presence of proteinase inhibitors the decrement of anion transport through band 3 was smaller indicating that removal achieved by proteinases includes oxidized band 3 which still retain transport ability. Proteinases recognize band 3 aggregates produced by peroxyl radicals as was evaluated by immunoblotting. It is concluded that decrease of band 3 transport capacity may result from a direct protein oxidation and from its degradation by proteinases and that band 3 aggregates removal may prevent macrophage recognition of the senescent condition which would lead to cell disposal.     

Peroxyl-oxidized Erythrocyte Membrane Band 3 Protein with Anion Transport Capacity is Degraded by Membrane-bound Proteinase

Human red blood cells anion exchange protein (band 3) exposed to peroxyl radicals produced by thermolysis of 2,2′-azo-bis(2-amidinopropane) (AAPH) is degraded by proteinases that prevent accumulation of oxidatively damaged proteins. To assess whether this degradation affects anion transport capacity we used the anionic fluorescent probe 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-y) amino] ethanosulfonate (NBD-taurine). A decrease of band 3 function was observed after exposure to peroxyl radicals. In the presence of proteinase inhibitors the decrement of anion transport through band 3 was smaller indicating that removal achieved by proteinases includes oxidized band 3 which still retain transport ability. Proteinases recognize band 3 aggregates produced by peroxyl radicals as was evaluated by immunoblotting. It is concluded that decrease of band 3 transport capacity may result from a direct protein oxidation and from its degradation by proteinases and that band 3 aggregates removal may prevent macrophage recognition of the senescent condition which would lead to cell disposal.     

华北低丘山地不同土地利用方式下土壤团聚体及其有机碳分布特征

土地利用变化影响土壤团聚性及有机碳分布,进而改变土壤碳循环过程。对太行山南部50年刺槐人工林（R50）、17年刺槐人工林（R17）、自然恢复林（NR）和农田（CL）等不同土地利用方式下的表层土壤（0-20 cm）进行了系统研究,利用湿筛法对土壤团聚体进行分级,并计算土壤结构稳定性参数（平均重量直径MWD,团聚体比例AR）及不同粒径团聚体有机碳贡献率,进而分析弃耕后土壤团聚体分布及团聚体有机碳含量变化。结果表明,土地利用方式对土壤团聚体粒径分布及团聚体有机碳含量有显著影响,自然恢复林与刺槐林的大团聚体（>0.25 mm）含量都高于农田,且自然恢复林的大团聚体增加更显著。MWD的计算结果表明：自然恢复林 > 刺槐人工林 > 农田,说明该区域的自然恢复方式更容易促进大团聚体的形成,并显著改良土壤结构及增强土壤团聚体稳定性。弃耕后,不同土地利用方式0-10 cm土层各粒径团聚体有机碳含量均高于农田,且团聚体有机碳含量与团聚体稳定性呈正相关。这些结果说明,研究区域的自然植被恢复和人工造林都可以显著提高土壤的固碳能力,且储存的有机碳主要存于大团聚体中,而农田的有机碳大都存于粘粒+粉粒团聚体中。自然植被恢复和人工造林均提高了土壤结构稳定性,是改善团粒结构、提高土壤质量的有效方式。     

Cryopreservation of pluripotent stem cell aggregates in defined protein‐free formulation

Cultivation of undifferentiated pluripotent stem cells (PSCs) as aggregates has emerged as an efficient culture configuration, enabling rapid and controlled large scale expansion. Aggregate‐based PSC cryopreservation facilitates the integrated process of cell expansion and cryopreservation, but its feasibility has not been demonstrated. The goals of current study are to assess the suitability of cryopreserving intact mouse embryonic stem cell (mESC) aggregates and investigate the effects of aggregate size and the formulation of cryopreservation solution on mESC survival and recovery. The results demonstrated the size‐dependent cell survival and recovery of intact aggregates. In particular, the generation of reactive oxygen species (ROS) and caspase activation were reduced for small aggregates (109 ± 55 μm) compared to medium (245 ± 77 μm) and large (365 ± 141 μm) ones, leading to the improved cell recovery. In addition, a defined protein‐free formulation was tested and found to promote the aggregate survival, eliminating the cell exposure to animal serum. The cryopreserved aggregates also maintained the pluripotent markers and the differentiation capacity into three‐germ layers after thawing. In summary, the cryopreservation of small PSC aggregates in a defined protein‐free formulation was shown to be a suitable approach toward a fully integrated expansion and cryopreservation process at large scale. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

Two-step growth curve from undisturbed cultures of Tetrahymena pyriformis

Single cells from developing two day granulocytic bone marrow colonies were transfered in agar cultures. After three to five days, 48 of 239 transfered single cells had transformed to single macrophages or proliferated to form aggregates of pure macrophages or mixed macrophage-granulocyte aggregates. Some granulocytes in colonies developing in vitro from bone marrow cells appear to have the capacity to transform to macrophages.